A novel moonlight function of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for immunomodulation.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an energy metabolism-related enzyme, which generates NADH in glycolysis. Our previous study revealed a novel role of exogenous GAPDH in the amelioration of lipopolysaccharide (LPS)-induced sepsis-related, severe acute lung injury (ALI) in mice. Here, we show the effect of extracellular GAPDH on the physiological functions of macrophages, which play an important role in the onset of sepsis and ALI. GAPDH has no effect on cell viability, while it strongly suppressed cell adhesion, spreading, and phagocytic function of LPS-stimulated macrophages. GAPDH treatment significantly reduced tumor necrosis factor (TNF)-α, while it induced interleukin (IL)-10 production from LPS-stimulated macrophages in a dose-dependent manner. It is noteworthy that heat inactivation of GAPDH lost its immunomodulatory activity. Correspondingly, NADH significantly inhibited TNF-α and enhanced IL-10 production with elevation of both M1/M2 macrophage markers. These data suggest that extracellular GAPDH induces intermediate M1/M2 macrophages for termination of inflammation, partly through its enzyme activity for generation of NADH. © 2018 BioFactors, 44(6):597-608, 2018.

Synergistic tumor suppression by a Perilla frutescens-derived methoxyflavanone and anti-cancer tyrosine kinase inhibitors in A549 human lung adenocarcinoma.

Anti-cancer tyrosine kinase inhibitors (TKIs) are effective in many types of cancers including non-small cell lung cancer, while appearance of TKI-resistant tumors suggests a need for the development of their potentiation strategies. We have previously shown that a methoxyflavanone derivative from the Asian medicinal herb Perilla frutescens (Perilla-derived methoxyflavanone; PDMF) shows a prominent anti-tumor activity against A549 human lung adenocarcinoma. Here we show that PDMF and anti-cancer TKIs (nilotinib, bosutinib, dasatinib, and ponatinib) synergistically suppress proliferation of A549 cells. Flow cytometric analysis indicated that co-stimulation with nilotinib (4 µM) and PDMF induced G2/M cell cycle arrest in low PDMF doses (10-50 µM), whereas this combination triggered de novo G1 arrest in higher PDMF dosages (50-125 µM). We also found that co-administration with nilotinib and PDMF significantly suppressed in vivo tumorigenicity of A549 cells in athymic nude mice.

Prominent IgE-binding and cytokine-inducing capacities of a newly cloned N-terminal region of Der f 14, an apolipophorin-like house dust mite allergen.

We previously characterized a 177-kDa allergen, M-177, from Dermatophagoides farinae. Thereafter, a counterpart to M-177 for Euroglyphus maynei was cloned as Eur m 14, and its sequence revealed that two environmental allergens, Mag 1 and Mag 3, are digested fragments of M-177. The aims of this study were to clone the cDNA of Der f 14 corresponding to M-177 and to elucidate the allergenic capacities of the N-terminal fragment of Der f 14 (Der f 14-N). Recombinant allergens were produced as trigger-factor-fused proteins in Escherichia coli. Der f 14-N showed the highest IgE-binding frequency among Der f 14-derived fragments in patients allergic to house dust mite by enzyme-linked immunosorbent assay. Der f 14-N showed the highest capacity to induce cell proliferation in murine lymphocyte and human peripheral mononuclear cells among Der f 14-derived fragments. Der f 14-N induced IL-13, IFN-γ and IL-17 production more than Der f 1 and Der f 2 in mouse, and induced IL-5 and IFN-γ production at levels comparable to those of Der f 1 and Der f 2 in some patients. The high prevalence of IgE binding to the Der f 14-N indicates that it could be an important mite allergen.

A methoxyflavanone derivative from the Asian medicinal herb (Perilla frutescens) induces p53-mediated G2/M cell cycle arrest and apoptosis in A549 human lung adenocarcinoma.

Perilla frutescens is an Asian dietary herb consumed as an essential seasoning in Japanese cuisine as well as used for a Chinese medicine. Here, we report that a newly found methoxyflavanone derivative from P. frutescens (Perilla-derived methoxyflavanone, PDMF; 8-hydroxy-5,7-dimethoxyflavanone) shows carcinostatic activity on human lung adenocarcinoma, A549. We found that treatment with PDMF significantly inhibited cell proliferation and decreased viability through induction of G2/M cell cycle arrest and apoptosis. The PDMF stimulation induces phosphorylation of tumor suppressor p53 on Ser15, and increases its protein amount in conjunction with up-regulation of downstream cyclin-dependent kinase inhibitor p21Cip1/Waf1 and proapoptotic caspases, caspase-9 and caspase-3. We also found that small interfering RNA knockdown of p53 completely abolished the PDMF-induced G2/M cell cycle arrest, and substantially abrogated its proapoptotic potency. These results suggest that PDMF represents a useful tumor-preventive phytochemical that triggers p53-driven G2/M cell cycle arrest and apoptosis.

Plasma Cluster Ions Reduce the IgE-Binding Capacity of House Dust Mite Allergens under a Simulated Indoor Environmental Condition.

BACKGROUND: A high level of house dust mite (HDM) allergens in a living environment is a risk factor for both sensitization to these allergens and asthmatic attacks. We previously showed that plasma cluster ions (PCIs) impaired the IgE-binding capacity of atomized crude allergens prepared from Japanese cedar pollen and fungus under experimental conditions. OBJECTIVE: We evaluated the capacity of PCIs to impair the IgE-binding capacity of airborne HDM allergens under a simulated indoor environmental condition. METHODS: For the determination of the effects of PCIs on HDM allergens under an experimental condition, HDM extract was atomized as aqueous mist into a cylindrical experimental apparatus filled with PCIs. For the evaluation of the effects of PCIs under a simulated natural indoor environmental condition, dried HDM allergens were floated as airborne particles in an acryl cubic apparatus in the presence of PCIs. The IgE-binding capacities of the PCI- and sham-treated HDM allergens were analyzed by an ELISA. RESULTS: The IgE-binding capacity of the HDM allergens was significantly impaired after PCI treatment compared to that after sham treatment under both experimental and simulated environmental conditions. The ELISA results demonstrated that the IgE-binding capacities of HDM allergens after PCI treatment showed 68 and 74% reductions compared to those after sham treatment under the experimental and simulated environmental conditions, respectively. CONCLUSIONS: PCIs have the capacity to impair the IgE-binding capacity of airborne HDM allergens in a simulated environmental condition.

A flavanone derivative from the Asian medicinal herb (Perilla frutescens) potently suppresses IgE-mediated immediate hypersensitivity reactions.

Perilla frutescens is a dietary leafy herb consumed as a traditional Japanese condiment as well as used for Chinese medicine with anti-inflammatory activity. Here we report a hitherto-unrecognized P. frutescens phytochemical that potently suppresses IgE-mediated type I hypersensitivity reactions. Structural analysis reveals that the purified anti-allergic compound (Perilla-derived methoxyflavanone, PDMF) is identified as 8-hydroxy-5,7-dimethoxyflavanone. PDMF significantly inhibits IgE-mediated histamine release from RBL-2H3 rat basophilic leukemia cells as compared with those seen in known P. frutescens-derived anti-inflammatory polyphenols. We also show that oral administration of PDMF not only suppresses passive cutaneous anaphylaxis, but also prevents allergic rhinitis-like nasal symptoms in a murine model of Japanese cedar pollinosis. Mechanistically, PDMF negatively regulates Akt phosphorylation and intracellular Ca2+ influx, both of which are essential for mast cell secretory granule translocation and its exocytosis upon high-affinity IgE receptor (FcεRI) cross-linking. These results represent PDMF as a new potent anti-allergic phytochemical useful for prevention of IgE-driven hypersensitivity reactions.

Exposure to positively- and negatively-charged plasma cluster ions impairs IgE-binding capacity of indoor cat and fungal allergens.

BACKGROUND: Environmental control to reduce the amount of allergens in a living place is thought to be important to avoid sensitization to airborne allergens. However, efficacy of environmental control on inactivation of airborne allergens is not fully investigated. We have previously reported that positively- and negatively-charged plasma cluster ions (PC-ions) reduce the IgE-binding capacity of crude allergens from Japanese cedar pollen as important seasonal airborne allergens. Cat (Felis domesticus) and fungus (Aspergillus fumigatus) are also important sources of common airborne allergens in living spaces throughout the year, and early sensitization with those allergens is considered to be a risk factor for future development of allergic rhinitis, pollinosis and asthma. The aim of this study is to examine whether the PC-ions reduce the IgE-binding capacity of a cat major allergen (Fel d 1) and fungal allergens in an experimental condition. METHODS: Fel d 1, crude fungal extract, or a fungal major allergen Asp f 1, was treated with PC-ions for 6 h in an experimental cylindrical apparatus. Sham-treated allergens were prepared in the same experimental apparatus without generation of PC-ions. The degradation of the PC-ions-treated Fel d 1 was analyzed by SDS-PAGE, and the IgE-binding capacity of the PC-ions-treated allergens was analyzed by ELISA inhibition assay. RESULTS: Exposure of Fel d 1, crude fungal extract and Asp f 1 to PC-ions significantly decreased protein content of Fel d 1 or Asp f 1, respectively. SDS-PAGE analysis suggested that the decreased Fel d 1 content upon exposure with PC-ions was attributable to protein degradation. ELISA inhibition indicated that the PC-ions treatment significantly impaired IgE-binding capacities of Fel d 1, crude fungal allergens, and Asp f 1 compared to sham treatment. DISCUSSION: Our data suggest that treatment with PC-ions not only reduce indoor cat and fungal allergens, but also impair their allergenicity. CONCLUSION: These results suggest that environmental control with PC-ions is useful for inactivation of indoor cat and fungal allergens.

Der f 34, a Novel Major House Dust Mite Allergen Belonging to a Highly Conserved Rid/YjgF/YER057c/UK114 Family of Imine Deaminases.

The high prevalence of house dust mite (HDM) allergy is a growing health problem worldwide, and the characterization of clinically important HDM allergens is a prerequisite for the development of diagnostic and therapeutic strategies. Here, we report a novel HDM allergen that belongs structurally to the highly conserved Rid/YjgF/YER057c/UK114 family (Rid family) with imine deaminase activity. Isolated HDM cDNA, named der f 34, encodes 128 amino acids homologous to Rid-like proteins. This new protein belongs to the Rid family and has seven conserved residues involved in enamine/imine deaminase activity. Indeed, we demonstrated that purified Der f 34 had imine deaminase activity that preferentially acted on leucine and methionine. Native Der f 34 showed a high IgE binding frequency as revealed by two-dimensional immunoblotting (62.5%) or ELISA (68%), which was comparable with those of a major HDM allergen Der f 2 (77.5 and 79%, respectively). We also found that Der f 34 showed cross-reactivity with another prominent indoor allergen source, Aspergillus fumigatus This is the first report showing that the Rid family imine deaminase represents an additional important pan-allergen that is conserved across organisms.

Impact of Histone H1 on the Progression of Allergic Rhinitis and Its Suppression by Neutralizing Antibody in Mice.

Nuclear antigens are known to trigger off innate and adaptive immune responses. Recent studies have found that the complex of nucleic acids and core histones that are derived from damaged cells may regulate allergic responses. However, no fundamental study has been performed concerning the role of linker histone H1 in mast cell-mediated type I hyperreactivity. In this study, we explored the impact of histone H1 on mast cell-mediated allergic responses both in vitro and in vivo. In the course of a bona-fide experimental allergen sensitization model upon co-injection with alum adjuvant, ovalbumin (OVA), but not PBS, induced elevated levels of circulating histone H1. Intranasal challenge with histone H1 to OVA/alum- (but not PBS/alum)-sensitized mice induced significantly severer symptoms of allergic rhinitis than those in mice sensitized and challenged with OVA. A monoclonal antibody against histone H1 not only suppressed mast cell degranulation, but also ameliorated OVA-induced nasal hyperreactivity and IgE-mediated passive cutaneous anaphylaxis. Our present data suggest that nuclear histone H1 represents an alarmin-like endogenous mediator acting on mast cells, and that its blockage has a therapeutic potential for mast cell-mediated type I hyperreactivity.

Identification of amino acid residues that determine the substrate specificity of mammalian membrane-bound front-end fatty acid desaturases.

Membrane-bound desaturases are physiologically and industrially important enzymes that are involved in the production of diverse fatty acids such as polyunsaturated fatty acids and their derivatives. Here, we identified amino acid residues that determine the substrate specificity of rat Δ6 desaturase (D6d) acting on linoleoyl-CoA by comparing its amino acid sequence with that of Δ5 desaturase (D5d), which converts dihomo-γ-linolenoyl-CoA. The N-terminal cytochrome b5-like domain was excluded as a determinant by domain swapping analysis. Substitution of eight amino acid residues (Ser209, Asn211, Arg216, Ser235, Leu236, Trp244, Gln245, and Val344) of D6d with the corresponding residues of D5d by site-directed mutagenesis switched the substrate specificity from linoleoyl-CoA to dihomo-γ-linolenoyl-CoA. In addition, replacement of Leu323 of D6d with Phe323 on the basis of the amino acid sequence of zebra fish Δ5/6 bifunctional desaturase was found to render D6d bifunctional. Homology modeling of D6d using recent crystal structure data of human stearoyl-CoA (Δ9) desaturase revealed that Arg216, Trp244, Gln245, and Leu323 are located near the substrate-binding pocket. To our knowledge, this is the first report on the structural basis of the substrate specificity of a mammalian front-end fatty acid desaturase, which will aid in efficient production of value-added fatty acids.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) prevents lipopolysaccharide (LPS)-induced, sepsis-related severe acute lung injury in mice.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an energy metabolism-related enzyme in the glycolytic pathway. Recently, it has been reported that GAPDH has other physiological functions, such as apoptosis, DNA repair and autophagy. Some in vitro studies have indicated immunological aspects of GAPDH function, although there is no definite study discussing the advantage of GAPDH as a therapeutic target. Here, we show that GAPDH has an anti-inflammatory function by using a lipopolysaccharide (LPS)-induced, sepsis-related severe acute lung injury (ALI) mouse model, which is referred to as acute respiratory distress syndrome (ARDS) in humans. GAPDH pre-injected mice were protected from septic death, and their serum levels of proinflammatory cytokines were significantly suppressed. In lung tissue, LPS-induced acute injury and neutrophil accumulation were strongly inhibited by GAPDH pre-injection. Pulmonary, proinflammatory cytokine gene expression and serum chemokine expression in GAPDH pre-injected mice were also reduced. These data suggest the therapeutic potential of GAPDH for sepsis-related ALI/ARDS.

Regulation of polyunsaturated fatty acid biosynthesis by seaweed fucoxanthin and its metabolite in cultured hepatocytes.

The effects of a seaweed carotenoid, fucoxanthin, and its physiological metabolite, fucoxanthinol, on the biosynthesis of polyunsaturated fatty acids (PUFA) were investigated using cultured rat hepatoma BRL-3A. The metabolism of α-linolenic acid (18:3n-3) was suppressed by the addition of these carotenoids, resulting in a decrease in the content of eicosapentaenoic acid (20:5n-3), which suggested a down-regulation of metabolic enzymes such as fatty acid desaturase and elongase. An increase in the content of docosahexaenoic acid (22:6n-3), as observed in previous studies in vivo, might be a buffering action to maintain the membrane fluidity. The suppressive effect of fucoxanthinol on ∆6 fatty acid desaturase was not at the level of gene expression but due to specific modifications of the protein via a ubiquitin-proteasome system. A proteomic analysis revealed several factors such as phosphatidylethanolamine-binding protein that might be involved in the observed action of fucoxanthin. These findings will contribute to studies on the elucidation of the precise molecular mechanisms underlying the regulation of PUFA biosynthesis by fucoxanthin.

Utilization of waste syrup for production of polyunsaturated fatty acids and xanthophylls by Aurantiochytrium.

In the food industry, syrups containing a high concentration of sugar used for fruit preservation is abundantly discharged as a food processing waste and disposed by incineration, resulting in the rise of the manufacturing cost and environmental pollution. This study demonstrates how waste syrup can be utilized as carbon source for production of docosahexaenoic acid (DHA) and astaxanthin by the thraustochytrid strain, Aurantiochytrium sp. KH105. The strain could grow in culture medium containing 3-50% waste syrup, and the maximum yields of DHA and astaxanthin were 207.6 mg/L (at 50%) and 1.1 mg/L (at 25%), respectively. After the optimization of culture medium composition by response surface method, DHA and astaxanthin yields increased by 2.1 and 1.5 fold, respectively. When the waste syrup was treated with activated charcoal, citrate concentration in the syrup was reduced and the astaxanthin yield increased by 2.3 fold. This study shows that the waste syrup can be effectively used for the functional lipid production by the thraustochytrid.

Competitive advantage and tolerance of selected shochu yeast in barley shochu mash.

A shochu yeast strain, Saccharomyces cerevisiae BAW-6, was previously isolated from Kagoshima yeast strain Ko, and has since been utilized in shochu production. The BAW-6 strain carries pho3/pho3 homozygous genes in contrast to the heterozygous PHO3/pho3 genes in the parental Ko strain. However, absence of the PHO3 gene per se cannot explain the fermentation superiority of BAW-6. Here, we demonstrate the growth advantage of the BAW-6 strain over the Ko strain by competitive cultivation in barley shochu preparation, where alcohol yield and nihonshudo of the former strain were higher than those of the latter strain. In addition, the maximum growth rate of BAW-6 was less affected than that of Ko by high Brix values of barley koji medium, suggesting that BAW-6 is less sensitive to growth inhibitory compounds derived from barley or barley koji. The tolerance of BAW-6 to growth inhibitory compounds, cerulenin and diethylstilbestrol (an H⁺-ATPase inhibitor), was also higher than that of other yeast strains. Consistent with BAW-6's tolerance to diethylstilbestrol in the presence of 8% ethanol (pH 4.5), H⁺-ATPase activity, but not transcription of its gene, was higher in BAW-6 than in Ko. We conclude that the BAW-6 strain is associated with certain gene alterations other than PHO3, such that it can maintain cellular ion homeostasis under conditions of ethanol stress during the latter phase of fermentation.

Genetic instability of constitutive acid phosphatase in shochu and sake yeast.

Genetic instability of constitutive acid phosphatase (cAPase) activity was observed in a shochu brewer's yeast strain (Ko), which consistently produced 0.3-1% progeny without cAPase when it had been subcultured for a long period of time in barley shochu mash or in conventional complete medium. Genetic analysis showed that the cAPase-negative phenotype was associated with a single mutation in the PHO3 gene and that the Ko strain had heteroallelic PHO3/pho3 genes, while the PHO3⁻ mutants had the homoallelic pho3/pho3 defect. Some sake yeast strains that are cAPase negative, such as K6, K7 and K9, also had the same homoallelic defect, whereas another sake yeast strain K3, with heteroallelic PHO3/pho3 genes, displayed similar genetic instability of cAPase activity. In all cases, the pho3-defective genes were generated by deletion of an approximately 1.9 kb region between the PHO5-PHO3 tandem genes on chromosome II, resulting in chimeric PHO5/3 fusion genes with different fusion points. By integrating a lys2 marker, which is linked with the pho3 allele on the arm of chromosome II in the Ko strain, we demonstrated that the pho3/pho3 defect originated either from a loss of heterozygosity at the heteroallelic PHO3/pho3 locus or from a looping out of the PHO3 region. Although fermentation experiments have not yet indicated any correlation between cAPase activity and alcohol production, the PHO3⁻ mutation itself could prove to be a useful selective marker for yeast strains carrying a number of advantageous mutations for fermentation and which display phenotypic diversity and stability.

Unexpected T cell regulatory activity of anti-histone H1 autoantibody: its mode of action in regulatory T cell-dependent and -independent manners.

Induction of anti-nuclear antibodies against DNA or histones is a hallmark of autoimmune disorders, but their actual contribution to disease predisposition remains to be clarified. We have previously reported that autoantibodies against histone H1 work as a critical graft survival factor in a rat model of tolerogeneic liver transplantation. Here we show that an immunosuppressive anti-histone H1 monoclonal antibody (anti-H1 mAb) acts directly on T cells to inhibit their activation in response to T cell receptor (TCR) ligation. Intriguingly, the T cell activation inhibitory activity of anti-H1 mAb under suboptimal dosages required regulatory T (Treg) cells, while high dose stimulation with anti-H1 mAb triggered a Treg cell-independent, direct negative regulation of T cell activation upon TCR cross-linking. In the Treg cell-dependent mode of immunosuppressive action, anti-H1 mAb did not induce the expansion of CD4(+-)Foxp3(+) Treg cells, but rather potentiated their regulatory capacity. These results reveal a previously unappreciated T cell regulatory role of anti-H1 autoantibody, whose overproduction is generally thought to be pathogenic in the autoimmune settings.

Immunological and regenerative aspects of hepatic mast cells in liver allograft rejection and tolerance.

The precise roles of mast cells in liver allograft rejection and tolerance are still unknown. This study aimed to explore the roles of mast cells in immune regulation and liver regeneration for tolerance induction by using rat models of orthotopic liver transplantation (OLT). Stem cell factor (SCF) and its receptor c-Kit, which are critical to the migration and development of not only stem cells but also mast cells, significantly increased in the tolerogenic livers as compared with rejected livers. The significant elevation of mast cell tryptase, high-affinity IgE receptor, and histamine suggested the activation of mast cells in liver allografts at the tolerogenic phase after OLT. Immunohistochemical analysis using confocal microscope clearly showed colocalization of mast cells, Foxp3+ Tregs, γδ T cells, and recipient-derived hepatic progenitor cells with higher expression of SCF, IL-9, IL-10, TGF-ß1, and IL-17 related to immunoregulation and liver regeneration in the donor grafts of a tolerogenic OLT model. Cross-talk among mast cells and other cells was evaluated by in vitro studies demonstrating that syngeneic bone marrow-derived mast cells (BMMCs) co-cultured with naïve splenocytes or primary hepatocytes significantly increased the population of splenic γδ T cells by mitogen stimulation or by mast cell degranulation, and also significantly induced the hepatocyte proliferation, respectively. Our results suggested that mast cells in the donor grafts may play important roles in the induction/maintenance of immune tolerance and liver regeneration resulting in the replacement of hepatic cells from donor to recipient.

Prophylactic effect of Lactobacillus oral vaccine expressing a Japanese cedar pollen allergen.

Lactic acid bacteria (LAB) represent an attractive delivery vehicle for oral allergy vaccine because of their safety as a food microorganism as well as their potent adjuvant activity triggering anti-allergic immune response. Here, we report the generation of recombinant LAB expressing a major Japanese cedar pollen allergen Cry j 1 (Cry j 1-LAB), and their prophylactic effect in vivo. To facilitate heterologous expression, the codon usage in the Cry j 1 gene was optimized for the host LAB strain Lactobacillus plantarum by the recursive PCR-based exhaustive site-directed mutagenesis. Use of the codon-optimized Cry j 1 cDNA and a lactate dehydrogenase gene fusion system led to a successful production of recombinant Cry j 1 in L. plantarum NCL21. We also found that oral vaccination with the Cry j 1-LAB suppressed allergen-specific IgE response and nasal symptoms in a murine model of cedar pollinosis.

Sake lees fermented with lactic acid bacteria prevents allergic rhinitis-like symptoms and IgE-mediated basophil degranulation.

We tested the effect of oral administration of fermented sake lees with lactic acid bacteria (FESLAB) on a murine model of allergic rhinitis upon immunization and nasal sensitization with ovalbumin (OVA). We used Lactobacillus paracasei NPSRIk-4 (isolated from sake lees), and L. brevis NPSRIv-8 (from fermented milk) as starter strains to produce the FESLAB. Oral FESLAB administration resulted in the development of significantly fewer sneezing symptoms than those seen in sham control animals given sterile water. We also found that FESLAB suppressed the allergen-induced degranulation of RBL2H3 rat basophilic leukemia cells.

Purification and characterization of intracellular lipase from the polyunsaturated fatty acid-producing fungus Mortierella alliacea.

Previous studies on an arachidonic acid-producing fungus, Mortierella alliacea YN-15, suggested that its intracellular lipase plays an important role in the metabolism of exogenous and storage lipids. The lipase purified in this study through acetone precipitation and three-step chromatography was estimated to be about 11 kDa in size by SDS-PAGE and mass spectrometry, and it tended to form large aggregates in aqueous solution. The purified lipase retained its activity over wide ranges of pH (2-12) and temperature (20-80 °C). Its activity was enhanced by the Ca(2+) ion and reduced by some heavy metal ions, such as Zn(2+) and Hg(2+), and diethylpyrocarbonate. Among the various substrates tested, monoacylglycerols containing long-chain unsaturated fatty acids and phosphatidylcholine were preferentially hydrolyzed over triacylglycerols and fatty acid methyl esters. The lipase strongly hydrolyzed the sn-1/3 ester bonds and weakly hydrolyzed the sn-2 ester bonds of triolein, and it also catalyzed the acylglycerol synthesis reaction in a solvent-free two-phase system. The results indicate that triacylglycerol may be formed via 2-monoacylglycerol. Thus, the highly stable M. alliacea lipase may be useful for the synthesis of structured lipids, particularly acylglycerols containing functional unsaturated fatty acids at the sn-2 position.